Female Infertility and Antioxidants

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Female Infertility and Antioxidants
Lucky H. Sekhon, Sajal Gupta, Yesul Kim and Ashok Agarwal*
Center for Reproductive Medicine, Glickman Urological & Kidney Institute and Ob/Gyn& Women’s Health Institute,
Cleveland Clinic 9500 Euclid Avenue, Desk A19.1, Cleveland, OH 44195, USA
Abstract: Aim: Many studies have implicated oxidative stress in the pathogenesis of infertility causing diseases of the fe-
male reproductive tract. The aim of this study was to review the current literature on the effects of antioxidant therapy and
to elucidate whether antioxidant supplementation is useful to prevent and/or treat infertility and poor pregnancy
outcomes related to various obstetric and gynecologic conditions.
Methods: Review of recent publications through Pubmed and the Cochrane data base.
Results: Antioxidant supplementation has been shown to improve insulin sensitivity and restore redox balance in patients
with PCOS. Supplementation with RU486, Curcuma longa, melatonin, caffeic acid phenethyl ester (CAPE) and catechins
may induce remission and halt disease progression in endometriosis. Selenium therapy may improve pregnancy rates
in unexplained infertility. Currently there is no evidence to substantiate the use of antioxidants to prevent or treat
preeclampsia. Up to 50-60% of recurrent pregnancy loss may be attributable to oxidative stress. Observational studies
have confirmed a link between antioxidant-poor diet and recurrent pregnancy loss.
Conclusion: Although many advances are being made in the field of antioxidants therapy, there is a need for further
investigation using randomized controlled trials within a larger population to determine the efficacy and safety of
antioxidant supplementation.
INTRODUCTION
Reactive oxygen species (ROS) can modulate cellular functions, and oxidative stress (OS) can impair the intracel- lular milieu, resulting in diseased cells or endangered cell survival. Reproductive cells and tissues remain stable when free radical production and the scavenging antioxidants re- main in balance. The role of ROS in various diseases of the female reproductive tract has been investigated. ROS can affect a variety of physiological functions in the reproductive tract, and excessive levels can result in precipitous patholo- gies affecting female reproduction. The oxidant status can influence early embryo development by modifying the key transcription factors, hence modifying gene expression.
The review will focus on ROS homeostasis and genera- tion of OS in the female reproductive processes. Our review elucidates the role of ROS in physiological processes such as folliculogenesis, oocyte maturation, endometrial cycle, lute- olysis, implantation, and embryogenesis and the role of anti- oxidants in various reproductive pathologies. This review encapsulates the role of OS, which is becoming increasingly important as new evidence of its role in conditions such as polycystic ovarian disease and abortions is discovered. The review highlights how OS modulates natural and assisted
*Address correspondence to this author at the Professor & Director, Center
for Reproductive Medicine, Glickman Urological & Kidney Institute and
Ob/Gyn & Women’s Health Institute, Cleveland Clinic, 9500 Euclid Avenue, Desk A19.1, Cleveland, OH 44195, USA; Tel: (216) 444-9485;
Fax: (216) 445-6049; E-mail: [email protected]
fertility and the importance of antioxidant strategies to inter- cept OS to overcome its adverse effects.
WHAT IS OXIDATIVE STRESS?
Oxidative stress arises from an imbalance between pro- oxidant molecules generated from aerobic metabolism and
protective antioxidants. OS influences the entire reproduc-
tive lifespan of a woman. Reactive oxygen species may act as key signalling molecules in physiological processes but
at excess, uncontrolled levels they may also mediate patho-
logical processes involving the female reproductive tract. There is a body of literature providing clinical evidence that
substantiates the link between OS and female infertility.
Pro-Oxidants
prised of stable bonds formed by paired electrons. Weak-
ened, disrupted bonds allow for the generation of free radi- cals- unstable and highly reactive species with one or more
unpaired electrons. They gain stability by acquiring electrons
from nearby nucleic acids, lipids, proteins, and carbohy- drates, initiating a cascade of chain reactions that may result
in cellular damage and disease [1-4].
Reactive oxygen species are formed endogenously during aerobic metabolism and as a result of various metabolic pathways of oocytes and embryos or as part of the body’s defense mechanisms. ROS also can arise from exogenous sources, such as alcohol, tobacco, and various environmental
Female Infertility and Antioxidants Current Women’s Health Reviews, 2010, Vol. 6, No. 2 85
pollutants. ROS include hydroxyl radicals, superoxide anion, hydrogen peroxide, and nitric oxide (NO) [5]. Several bio- markers indicative of redox status, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), lipid peroxides, and nitric oxide, have been identified within the ovary, endometrium, fallopian tubes, embryo, placenta, and the peritoneal fluid of women. At controlled levels, free radicals are capable of exerting physiological effects and mediating processes such as tissue remodelling, hormone signalling, oocyte maturation, folliculogenesis, tubal func- tion, ovarian steroidogenesis, cyclical endometrial changes, and germ cell function [6, 7]. However, when ROS increase to pathological levels they are capable of inflicting signifi- cant damage to cell structures.
Antioxidants
repair of ROS-induced damage to cell structures [8]. Non-
enzymatic antioxidants include vitamin C, vitamin E, sele-
nium, zinc, beta carotene, carotene, taurine, hypotaurine,
cysteamine, and glutathione. Enzymatic antioxidants include
SOD, catalase, GSH-Px, glutaredoxin and glutathione reduc-
tase [5]. The degree of antioxidant defense present is often expressed as total antioxidant capacity (TAC) [6].
A disruption in the delicate balance between antioxidants
and pro-oxidant molecules can result in OS. OS arises when
the generation of reactive oxygen species and other radical
species overrides the scavenging capacity by antioxidants,
either due to the excessive production of ROS or an inade-
quate availability of antioxidants. Thus, oral antioxidant
supplementation may serve to prevent and alleviate OS and
its contribution to the pathogenesis of obstetrical disease
such as preeclampsia and recurrent pregnancy loss and gyne-
cological disorders such as polycystic ovarian syndrome (PCOS) and endometriosis.
OS IN THE FEMALE REPRODUCTIVE TRACT –
PHYSIOLOGICAL ROLE OF OS
demonstrated in normally cycling ovaries [9, 10]. The
follicular fluid microenvironment contains leukocytes,
macrophages, and cytokines, all of which are known sources
of ROS. ROS within the follicular fluid plays a role in
modulating oocyte maturation, folliculogenesis, ovarian
steroidogenesis, and luteolysis [11]. Follicular development
involves the progression of small primordial follicles into
large pre-ovulatory follicles. Studies have implicated the
nitric oxide radical in the follicular growth and programmed
follicular cell death that occur during folliculogenesis [12,
13]. Moderate OS levels are required for ovulation. The final
stages of oocyte maturation are associated with fluctuations
in cytokines, prostaglandins, proteolytic enzymes, nitric
oxide, and steroids, which increase the level of ROS,
influencing ovarian blood flow and eventually facilitating
follicle rupture [14]. A degree of oxidative enzyme activity
is exhibited by thecal cells, granulosa lutein cells, and hilus
cells, illustrating the role of OS in ovarian steroidogenesis [11].
ROS is controlled and kept at physiological levels within
the ovary by various antioxidant systems, including catalase,
vitamin E, glutathione and carotenoids [4]. SOD, a metal-
containing enzymatic antioxidant that catalyzes the
decomposition of superoxide into hydrogen peroxide and
oxygen, has been characterized in the theca interna cells in
the antral follicles. Therefore, the theca interna cells may
protect the oocyte from excess ROS during its maturation
[15]. Another antioxidant factor important for healthy
follicle development is transferrin, an iron-chelating glyco-
protein that suppresses ROS generation [16]. Vitamin C also
is known to have a protective effect within the follicle as
vitamin C deficiency has been reported to result in ovarian
atrophy, extensive follicular atresia, and premature resump- tion of meiosis [15].
The overall ROS scavenging ability of antioxidants
within the follicular fluid microenvironment may diminish
with reproductive aging. Carbone et al. demonstrated
decreased levels of follicular fluid catalase and SOD in older
women, whose oocytes were seen to exhibit lower
fertilization rates and decreased blastocyst development
compared with oocytes of younger women [17]. Therefore,
the redox status of the follicle is closely related to oocyte quality and fertilization capacity.
Endometrium
endometrium is well-established. OS-promoting alterations
in ROS and SOD levels have been demonstrated just prior to
menstruation, during the late-secretory phase [18]. Estrogen
and progesterone withdrawal in endometrial cells in vitro has
been associated with a decrease in SOD activity, resulting in
the unopposed activity of ROS [18]. Elevated lipid peroxide
and decreased SOD in the endometrium during the late-
secretory phase may modulate endometrial breakdown,
leading to menstruation. NO is known to regulate the
endometrial microvasculature and is produced by endothelial
NO synthase (NOS), which is distributed in the glandular
surface epithelial cells of the endometrium [19]. NO is
thought to mediate endometrial decidualization and
menstruation as endothelial NOS mRNA expression has
been detected in the mid-secretory and late-secretory phase.
Endothelial NOS is also implicated in the changes seen in
the endometrium in preparation for implantation [20]. ROS
may mediate the physiological processes of shedding and
implantation by its activation of nuclear factor B within
the endometrium, leading to increased cyclooxygenase-2 mRNA and prostaglandin F2 synthesis [18].
Infertility
Approximately 1.3 million American couples receive medical advice or treatment for infertility every year [21]. Infertility is a disease defined as the inability to conceive following 12 or more months of unprotected sex [22]. In general, an estimated 84% of couples conceive after 1 year of intercourse, and 92% of the couples conceive after 2 years
86 Current Women’s Health Reviews, 2010, Vol. 6, No. 2 Sekhon et al.
[23]. A primary diagnosis of male factor infertility is made in 30% of infertile couples. High levels of ROS biomarkers have been detected in semen samples of 25-40% of infertile men [5]. Although ROS have a physiological role in normal sperm function, mediating the acrosome reaction, hyperacti- vation, motility, and capacitation of spermatozoa, excessive levels of ROS may arise from immotile or morphologically abnormal spermatozoa and leukocytes. Spermatozoa lack the necessary cytoplasmic antioxidant enzymes and are vulnerable to OS-induced DNA damage and apoptosis [5, 24]. Substantial evidence exists that implicates OS in many causes of male infertility. Oral antioxidant supple- mentation has become standard practice for male infertility [5].
Combined female and male factor infertility is responsi- ble for 20%–30% of cases. If the results of a standard infer- tility examination are normal, a diagnosis of unexplained or idiopathic infertility is assigned [25]. OS has a well- established role in pathogenesis of unexplained infertility, which is seen to affect 15% of couples [25]. Although the frequency and origin of different forms of infertility varies, 40%–50% of the etiology of infertility studied is due to female causes [26].
OS induces infertility in women through a variety of mechanisms. Excess ROS in the follicle may overwhelm follicular fluid antioxidant defense and directly damage oo- cytes. The DNA of oocytes and spermatozoa may be dam- aged, leading to defective fertilization when the peritoneal cavity microenvironment is plagued with severe OS. Even when fertilization is achieved, OS-induced apoptosis may result in embryo fragmentation, implantation failure, abor- tion, impaired placentation, and congenital abnormalities [27]. Excess ROS may hinder the endometrium, which nor- mally functions to support the embryo and its development [28]. OS may induce luteal regression and insufficient luteal hormonal support for the continuation of a pregnancy [8]. The association of OS with various gynecologic and obstet- ric conditions related to infertility suggests a potential role
for oral antioxidant supplementation (Fig. 1). Additional research is needed to determine whether such supplementa- tion can ensure successful fertilization and pregnancy by controlling the OS experienced by patients with endometri- osis, PCOS, unexplained infertility, preeclampsia, and recur- rent pregnancy loss.
THE USE OF ANTIOXIDANTS IN TREATMENT OF
GYNECOLOGICAL CONDITIONS
Polycystic Ovarian Syndrome
PCOS is an anovulatory cause of infertility affecting 6- 10% of premenopausal women [29-32]. PCOS often can be characterized by hyperandrogenism, hirsutism, and oligo- menorrhea or amenorrhea. Metabolic, endocrinologic, and cardiovascular disorders may also coexist. Oxidative stress has been implicated in mediating the insulin resistance and increase in androgens seen in these patients [33].
A recent study by Kuscu et al. demonstrated increased MDA levels and upregulated SOD activity in patients with PCOS compared to controls. MDA levels were highest in patients who exhibited insulin resistance [34]. Insulin resistance and hyperglycemia are established as factors that increase oxidative stress. Fulghesu et al. evaluated the effect of N-acetyl-cysteine (NAC), known to replenish stores of the anti-oxidant glutathione, on insulin secretion and peripheral insulin resistance in subjects with PCOS. Patients were treated for 5-6 weeks with a 1.8g oral NAC per day. Massively obese patients were given a higher dose of 3g per day. NAC treatment was found to improve parameters of glucose control in hyperinsulinemic patients. Insulin levels were reduced, with increased peripheral insulin sensitivity. Therefore, the anti-oxidant effects of NAC may serve as a therapeutic strategy to improve the level of circulating insulin and insulin sensitivity in PCOS patients with hyperinsulinemia [35].
Non-obese PCOS patients without insulin resistance also have been reported to have elevated total oxidant and anti-
Fig. (1). The role of oxidative stress in obstetric and gynecologic conditions that contribute to infertility.
Female Infertility and Antioxidants Current Women’s Health Reviews, 2010, Vol. 6, No. 2 87
oxidant status [36]. Verit et al. demonstrated that total anti- oxidant status in these types of PCOS patients was correlated with raised luteinizing hormone levels and free androgen and dehydroepiandrosterone (DHEAS) levels [36]. Yilmaz et al. studied the effects of 12 weeks of treatment with oral hypoglycemic agents on OS in lean patients with PCOS [37]. Before treatment, PCOS patients exhibited OS with significantly raised serum MDA and homocysteine and significantly decreased serum TAS. PCOS patients treated with rosiglitazone showed an increase in TAS and a decrease in MDA levels, compared with a metformin-administered patient group in which these parameters did not change [37]. Therefore, rosiglitazone may be useful in combating OS in hyperinsulinemic PCOS patients.
Zhang et al. used methods of chemicalorimetry to measure and compare levels of serum lipid peroxides (LPO),
MDA, SOD, vitamin E, and vitamin C in patients with
PCOS and normal women [38]. Levels of serum LPO and MDA in patients with PCOS were significantly higher than
those found in normal women. Levels of vitamin E, vitamin
C, and SOD were lower in patients with PCOS than in the control group. After 3 months of therapy with oral
ethinylestradiol and cyproterone acetate tablets (Diane-35 ®
,
Merck, Whitehouse Station, N.J.), an anti-androgenic oral contraceptive often used to treat hirsutism associated with
PCOS, MDA and LPO levels decreased, while vitamin E,
vitamin C, and SOD levels increased in patients with PCOS [38]. Therefore, this therapy may alleviate the symptoms of
PCOS through both its anti-androgenic and anti-oxidant
actions.
Endometriosis
Severe cases of endometriosis are thought to render a
woman infertile by mechanical hindrance of the sperm-egg union by adhesions, endometriomata, and pelvic anatomy
malformations. However, in women with mild-to-moderate
forms of endometriosis and no pelvic anatomical distortion, the mechanism by which their fertility is reduced is poorly
understood.
ROS production may be amplified in the setting of en-
dometriosis due to menstrual reflux, which subjects the peri-
toneal cavity to pro-inflammatory hemoglobin and heme molecules released from transplanted erythrocyte debris.
Peritoneal fluid containing ROS-generating iron, macro-
phages, and environmental contaminants such as polychlori- nated biphenyls may disrupt the prooxidant/antioxidant bal-
ance, resulting in increased proliferation of tissue and adhe-
sions [39-42]. ROS are thought to promote the growth and adhesion of endometrial cells in the peritoneal cavity, con-
tributing to the pelvic anatomical distortion known to cause
infertility in endometriosis [43]. OS may have a role in pro- moting angiogenesis in ectopic endometrial implants by
increasing vascular endothelial growth factor (VEGF) produc-
tion [44]. This effect is partly mediated by glycodelin, a glycoprotein whose expression is stimulated by OS.
Glycodelin may act as an autocrine factor within ectopic
endometrial tissue by augmenting VEGF expression [44].
Altered molecular genetic pathways may contribute to the effects of OS in the pathogenesis of endometriosis and
endometriosis-associated infertility. Differential gene ex- pression of ectopic and normal endometrial tissue has been identified, including differential gene expression of glu- tathione-S-transferase, an enzyme in the metabolism of the potent antioxidant glutathione [45]. This suggests that altered molecular genetic pathways may determine the development of OS and its ability to induce cellular proliferation and an- giogenesis in women with endometriosis.
Peritoneal fluid of women with endometriosis has been
reported to exhibit increased ROS generation by activated
peritoneal macrophages [46]. Increased macrophage activity
is accompanied by the release of cytokines and other im-
mune mediators such as NO. NO is a pro-inflammatory free
radical that exerts deleterious effects on fertility by increas-
ing the amount of OS in the peritoneal fluid, an environment
that hosts the processes of ovulation, gamete transportation,
sperm-oocyte interaction, fertilization, and early embryonic
development [2, 47, 48]. However, the results of further
studies with large patient numbers failed to confirm an anti-
oxidant or oxidant imbalance as ROS levels in peritoneal
fluid of patients with endometriosis were not reported to be significantly higher than controls [49, 50].
After adjusting for confounding factors such as age,
BMI, gravidity, serum vitamin E, and serum lipid levels,
Jackson et al. reported a weak relationship of elevated levels
of thiobarbituric acid reactive substances (TBARS), an over-
all measure of OS, in women with endometriosis [51]. In-
creased NO production and lipid peroxidation have been
reported in the endometrium of women with endometriosis
[2, 52]. However, several studies failed to find significant
differences in the peritoneal fluid levels of NO, lipid perox-
ide, and ROS in women with and without endometriosis- associated infertility.
The failure of some studies to confirm alterations in peri-
toneal fluid NO, lipid peroxide and antioxidant status in
women with endometriosis may be explained by the fact that
OS may occur locally, without affecting total peritoneal fluid
ROS concentration. Also, markers of OS may be transient and not detected at the time endometriosis is diagnosed.
An imbalance between ROS and antioxidant levels may
play an important role in the pathogenesis of endometriosis-
associated infertility. Increased concentrations of oviductal
fluid ROS may adversely affect oocyte and spermatozoa
viability and the process of fertilization and embryo implan-
tation. Also, pro-inflammatory macrophages and activated
neutrophils in the oviductal fluid may significantly amplify
ROS production by endometriotic foci [43]. Increased ROS
production may inflict oxidative damage to the sperm plasma
and acrosomal membranes, resulting in a loss of motility and
decreased spermatozoal ability to bind and penetrate the oo-
cyte. The various possible consequences of OS-induced
DNA damage include failed fertilization, reduced embryo quality, pregnancy failure, and spontaneous abortion.
Modest levels of OS have been shown to induce the pro- liferation of endometrial stromal cells in vitro, which has been shown to be inhibited by antioxidants [53]. Several studies have shown that the peritoneal fluid of women with endometriosis-associated infertility have insufficient antioxi-
88 Current Women’s Health Reviews, 2010, Vol. 6, No. 2 Sekhon et al.
dant defense, with lower total antioxidant capacity (TAC) and significantly reduced SOD levels [2, 47, 54].
An early study used a simple rabbit model to demonstrate
the beneficial effect of antioxidant therapy in halting pro-
gression of the disease [55]. SOD and catalase were instilled
in the rabbit peritoneal cavity and were shown to signifi-
cantly reduce the formation of intraperitoneal adhesions at
endometriosis sites by blocking the toxic effects of the su-
peroxide anion and hydrogen peroxide radicals [55]. More
recently, RU486- a potent antiprogestational agent with anti-
oxidant activity, has been shown to decrease the proliferation
of epithelial and stromal cells in endometriosis [56].
Another drug being investigated for its potential use in
the treatment of endometriosis-associated infertility is pen-
toxifylline, a 3’,5’-nucleotide phosphodiesterase inhibitor.
Pentoxifylline has potent immunomodulatory properties and
has been shown to significantly reduce the embryotoxic ef-
fects of hydrogen peroxide [57]. Zhang et al. conducted a
recent randomized control trial in which pentoxifylline
treatment failed to demonstrate significant reduction in en-
dometriosis-associated symptoms such as…